阿拉丁的DBCO蛋白质标记试剂盒（5mg 规格）（D1510877）提供了一种便捷的方法，可使用二苄基环辛炔（DBCO）对含有胺基的蛋白质进行标记，用于环张力促进的叠氮-炔环加成反应（SPAAC）反应（图1）。该试剂盒中提供的DBCO-PEG5-NHS酯，是一种异型双功能交联剂，可与伯胺反应，形成共价连接的DBCO基团，该基团可进一步与叠氮标记的蛋白（单独提供）反应，生成多种生物偶联物。该试剂盒含有用于进行 10 次标记反应和后续纯化以除去过量 DBCO 试剂的必要材料。标记和纯化操作最快可在90分钟内完成。该试剂盒已针对标记分子量在20 kDa至150 kDa之间的蛋白质进行了优化。
图1 SPAAC示意图
阿拉丁DBCO蛋白标记试剂盒的特点：
- 多功能——标记体积为0.5–5 mL的蛋白质（20–150 kDa）
- 化学选择性——DBCO与叠氮化物选择性反应
- 异型双功能—可与胺和叠氮化物发生反应
- 稳定性——PEG链增强了水溶性并减少了非特异性吸附
- 便捷性——标记和纯化最快可在90分钟内完成，实际操作时间仅需30分钟
其他所需试剂
- 1. 变速台式离心机。
- 2. 5毫升离心管
- 3. 待标记的目标蛋白（不含BSA或任何载体蛋白）
- 4. PBS缓冲液（pH 7.2-7.4）。
- 5. 紫外-可见分光光度计。
注意事项
- 1. 纯化后的蛋白质应置于不含伯胺（例如，铵离子、Tris、甘氨酸、乙醇胺、三乙胺、谷胱甘肽）或咪唑的缓冲液中。所有这些物质都会显著抑制蛋白质标记。
- 2. 不纯的蛋白质或用牛血清白蛋白（BSA）或明胶稳定的蛋白质将无法被良好标记。
- 3. 交联剂对湿气敏感。打开前应将试剂管平衡至室温，以防止试剂管内凝结水分。使用试剂盒中提供的无水溶剂配制储备液后，可将其分装并在-20°C下储存，在此条件下可稳定保存两个月。在-80°C下储存可保存更长时间。
- 4. 离心脱盐柱的上样体积必须在2–4 mL之间，以确保最佳性能。超出此范围的上样体积可能导致蛋白质回收率降低或未偶联试剂去除不彻底。
- 5. 请勿重复使用离心脱盐柱。
使用说明
- 1. 制备蛋白样品
- 1.1 将目标蛋白溶解于不含胺、铵离子或叠氮化物的缓冲液（pH 7-9）中，因为这些物质会干扰标记反应。如有必要，在标记前将目标蛋白通过透析或缓冲液置换的方式更换到合适的缓冲液中，例如PBS（1X）。
- 1.2 在PBS溶液中制备浓度为0.5–5 mg/mL的蛋白样品。最终体积应≤4mL。
- 2. 计算DBCO-PEG5-NHS体积
- DBCO-PEG5-NHS的最佳用量取决于所需的标记程度。通常，较高的蛋白质浓度或表面具有更多可接触伯胺（例如赖氨酸残基）的蛋白质，需要较少的DBCO-PEG5-NHS即可达到所需的标记密度。关于DBCO:蛋白质比例的建议，请参见表1。
| 蛋白浓度范围 | 摩尔比 (DBCO:Protein) |
| 0.5-1.0 mg/mL | 40:1-20:1 |
| 1.0-5.0 mg/mL | 20:1-10:1 |
表1. DBCO-PEG5-NHS与目标蛋白的推荐摩尔比
2.1 计算摩尔比为X：1所需的DBCO-PEG5-NHS试剂毫摩尔数：
2.2 计算需添加到反应体系中的20mM DBCO-PEG5-NHS体积：
2.3 示例：对于2.5mL的2 mg/mL IgG（分子量 = 150,000 g/mol），摩尔比为20:1时，需向制备好的样品中加入33.5 μL的20 mM DBCO-PEG5-NHS试剂。
3. 蛋白标记步骤
3.1 将DBCO-PEG5-NHS酯平衡至室温。
3.2 使用前，配制交联剂。向DBCO-PEG5-NHS的试剂管中加入1mL DMSO，得到20 mM的储备液。涡旋或用移液器反复吹吸直至溶液均一。
3.3 量取计算体积的20 mM DBCO-PEG5-NHS试剂加入制备好的蛋白溶液中。
3.4 将反应混合物在室温下孵育1小时，或在冰上孵育2小时。
4. 脱盐柱预平衡
4.1 将一个空的离心柱放入一个 50mL 收集管中。
4.2 通过反复倒置试剂瓶重悬纯化树脂。然后取 14.0 mL悬液加入柱中，让树脂沉降。让柱缓冲液依靠重力从柱中排出。初始时，可能需要施加一些压力使最初的几滴缓冲液流出。以 1,000 × g 离心柱子 2 分钟，弃去储存缓冲液，将柱子放回同一个收集管中。
注意：纯化树脂以 20% 乙醇的悬液形式提供，树脂与乙醇的体积比为 3:1 (v/v)。
4.3 通过在树脂床顶部加入 10mL PBS，以 1,000 × g 离心 2 分钟来平衡柱子，弃去流出液，此步骤总共重复 3 次。此时柱子平衡完成。
注意：使用定角转子时，在柱子背对转子中心的一侧做标记。在后续所有离心步骤中，将柱子放入离心机时，标记面应背对转子中心。
5. 纯化DBCO标记的蛋白
5.1 将平衡好的离心柱转移到一个新的收集管中。
5.2 小心地将整个反应混合物（2–4mL）加到柱子的中心位置。
5.3 脱盐柱1000xg离心2分钟，收集管中的流出即为纯化后的DBCO标记的蛋白。
6. 测定DOL（可选）
偶联反应的效率可以通过测量蛋白质在280 nm处的吸光度和DBCO基团在其激发最大值（309 nm）处的吸光度来确定。
6.1 对于高浓度的样品，用PBS将少量纯化的偶联物进行稀释。
6.2 测量蛋白质在280 nm（A280）和DBCO基团在309 nm（A309）处的吸光度。
6.3 使用以下公式计算样品中蛋白质的浓度。
其中：
1. εprotein：蛋白质在280 nm处的摩尔消光系数。通常，IgG在280 nm处的摩尔消光系数为203,000 cm⁻¹M⁻¹，这也适用于IgA、IgD和IgE。
2. 0.90是DBCO在A280处贡献的校正因子。
6.4 使用以下公式计算标记程度（DOL）。
其中：
1. εprotein：蛋白质在280 nm处的摩尔消光系数。通常，IgG在280 nm处的摩尔消光系数为203,000 cm⁻¹M⁻¹，这也适用于IgA、IgD和IgE。
2. 0.90是DBCO在A280处贡献的校正因子。
6.4 使用以下公式计算标记程度（DOL）。
其中：
1. εDBCO：DBCO基团在309 nm处的摩尔消光系数；12,000 M⁻¹cm⁻¹
7. 将DBCO标记的蛋白质与叠氮化物偶联（可选）
7.1 在无叠氮化物的缓冲液（如PBS）中制备叠氮化物标记的偶联配偶体（需单独购买；货号 A1510878；A1510880；A1510881）。
7.2 将制备好的叠氮化物标记的偶联配偶体以1.5至10倍的摩尔过量加入到DBCO标记的蛋白质中。
7.3 将反应混合物在室温下孵育4–12小时。
7.4 根据您的应用需求，您可以直接使用偶联后的蛋白质，或通过尺寸排阻色谱柱和填料进一步纯化偶联蛋白。
Aladdin’s DBCO Protein Labeling Kit (5mg scale) (D1510877) provides a convenient method to label proteins with dibenzylcyclooctyne (DBCO) for use in strain‑promoted azide‑alkyne cycloaddition (SPAAC) reactions (Figure 1). The kit uses DBCO-PEG5-NHS ester, a heterobifunctional crosslinker that readily reacts with primary amines, forming covalently attached DBCO groups that can be further reacted with an azide‑labeled coupling partner to produce diverse bioconjugates. The kit includes reagents for DBCO‑labeling and purification of ten 2mL samples with a concentration of 0.5–5 mg/mL. Labeling and purification can be performed in as little as 90 minutes. The kit is optimized for labeling proteins with molecular weights between 20 kDa and 150 kDa.
Figure 1 Schematic of SPAAC
Aladdin’s DBCO Protein Labeling Kit features include:
- Versatile—label proteins (20–150 kDa) with volumes of 0.5–5 mL
- Chemoselective—DBCO reacts selectively with azides
- Heterobifunctional—reactive towards amine and azide
- Stability—The PEG chain enhances water solubility and reduces non-specific adsorption
- Convenient—labeling and purification can be completed in as little as 90 minutes with 30 min hands-on time
Required materials not supplied
1. Variable‑speed benchtop microcentrifuge
2. 5mL centrifuge tubes
3. Desired Protein for labeling (free of BSA or any carrier protein)
4. PBS buffer (pH 7.2-7.4).
5. UV‑Vis spectrophotometer
Matters needing attention
1. The purified proteins should be in a buffer that does not contain primary amines (for example, ammonium ions, Tris, glycine, ethanolamine, triethylamine, glutathione) or imidazole. All of these substances significantly inhibit protein labeling.
2. Impure proteins or proteins stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.
3. The crosslinker is moisture‑sensitive. Equilibrate the vial to room temperature before opening to prevent moisture condensation inside the vial. After preparing the stock solution with the anhydrous solvent provided in the kit, it can be aliquoted and stored at –20 °C, where it remains stable for two months. Storage at –80 °C allows for even longer preservation.
4. The sample loading volume for Spin Desalting Columns must be between 2-4mL to ensure optimal performance. Loading volumes outside this range may lead to lower protein recovery or inefficient removal of unconjugated reagents.
5. Do not reuse Spin Desalting Columns.
Instructions for Use
1. Prepare the protein sample
1.1 Prepare the target protein in a buffer (pH 7-9) free of amines, ammonium ions, or azide as these will interfere with the labeling reaction. If necessary, dialyze or exchange the target protein into an appropriate buffer, such as PBS (1X), before labeling.
1.2 Prepare the protein sample at a concentration of 0.5–5 mg/mL in PBS solution. The final volume should be ≤4mL.
2. Calculations
The optimal amount of DBCO-PEG5-NHS will depend on the desired degree of labeling. In general, higher protein concentrations or proteins with a greater number of accessible primary amines (e.g., lysine residues) on the surface will require less DBCO-PEG5-NHS to achieve the desired labeling density. For recommendations on DBCO:protein ratios, see Table 1.
| Protein concentration Range | Molar Ratio (DBCO:Protein) |
| 0.5-1.0 mg/mL | 40:1-20:1 |
| 1.0-5.0 mg/mL | 20:1-10:1 |
Table 1. Recommended molar ratio of DBCO-PEG5-NHS to target protein
2.1 Use the following formula to calculate the amount (in millimoles) of crosslinker to add to the sample for an X:1 molar ratio.
2.2 Use the following formula to calculate the volume (in μL) of 20 mM crosslinker to add to the sample.
2.3 For 2.5mL of 2 mg/mL IgG (MW = 150,000 g/mol) with 20:1 molar ratio, add 33.5 μL of 20 mM DBCO-NHS to the prepared sample.
3. Protein labeling procedure
3.1 Equilibrate DBCO-PEG5-NHS ester to room temperature.
3.2 Just before use, reconstitute the crosslinker. Add 1mL of DMSO to the vial of DBCO-PEG5-NHS to obtain a 20 mM stock solution. Vortex or pipette up and down until the solution is homogeneous.
3.3 Add the calculated volume of 20 mM DBCO-PEG5-NHS to the tube.
3.4 Incubate the reaction mixture for 1 hour at room temperature or 2 hours on ice.
4. Prepare the spin column
4.1 Place an empty spin column in a 50mL collection tube.
4.2 Suspend the purification resin by repeated inversion of the reagent bottle. Then add 14.0 mL of the suspension into the column and allow the resin to settle. Allow the column buffer to drain from the column by gravity. Initially, some pressure may be required to cause the first few drops of buffer to elute. Centrifuge the column at 1,000 × g for 2 minutes, discard the storage buffer and return column to the same collection tube.
Note: The purification resin is supplied as a slurry in 20% ethanol, with a resin-to-ethanol volume ratio of 3:1 (v/v).
4.3 Equilibrate the column by adding 5mL of PBS to the top of the resin bed and centrifuging at 1,000 × g for 2 minutes. Discard the flowthrough and repeat this step a total of 3 times.
Note: When using a fixed-angle rotor, place a mark on the side of the column that faces away from the rotor center. For all subsequent centrifugation steps, place the column in the microcentrifuge with the mark facing away from the rotor center.
5. Purify the DBCO‑labeled protein
5.1 Transfer the equilibrated column into a new collection tube.
5.2 Carefully pipette the entire reaction mixture (≤4mL) onto the center of the column.
Note: For samples < 2mL, add ultrapure water on top of the absorbed sample to increase protein recovery.
5.3 Centrifuge the column‑tube assembly at 1,000 × g for 2 minutes. The purified DBCO‑labeled protein is in the collection tube.
6. Determine the Degree of Labeling (optional)
The efficiency of the conjugation reaction can be determined by measuring the absorbance of the protein at 280 nm and the absorbance of the DBCO group at its excitation maximum (309 nm).
6.1 For samples with high concentrations, dilute a small amount of the purified conjugate with PBS.
6.2 Measure the absorbance of the protein at 280 nm (A280) and the DBCO group at 309 nm (A309).
6.3 Calculate the concentration of the protein in the sample using the following formula.
Where:
1. εprotein: Molar extinction coefficient of protein at 280 nm. Generally, the molar extinction coefficient of IgG at 280 nm is 203,000 cm⁻¹M⁻¹, and this also applies to IgA, IgD and IgE.
2. 0.90 is a correction factor for the DBCO contribution to A280.
6.4 Calculate the degree of labeling (DOL) using the following formula.
Where:
1. εDBCO: Molar extinction coefficient of the DBCO group at 309 nm is 12,000 M⁻¹cm⁻¹
7. Conjugate the DBCO‑labeled protein to an azide (optional)
7.1 Prepare the azide‑labeled coupling partner (supplied separately; Cat. No. A1510878; A1510880; A1510881) in an azide‑free buffer, such as PBS.
7.2 Add the prepared azide‑labeled coupling partner to the DCBO‑labeled protein at a 1.5 to 10 fold molar excess.
7.3 Incubate the reaction mixture for 4–12 hours at room temperature.
7.4 Depending on your application, you can use the conjugated protein immediately or purify the conjugated protein by size exclusion chromatography columns and resins.
ZJ26F0434369
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